The Cry3 class of Bacillus thuringiensis (Bt) Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis (Bt) are used in biopesticides and transgenic crops to control larvae of leaf feeding beetles and rootworms. Due to limited efficacy of Cry3-based biopesticides and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (Gelernter, 2004). Cry3Bb is toxic to corn rootworms (Donovan et al., 1992; Johnson et al., 1993), and a modified version is expressed in commercialized MON863 corn hybrids (Vaughn et al., 2005).
Cry3 toxins have a mode-of-action that is similar yet distinct from the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73-kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (Carroll et al., 1997; Loseva et al., 2002). Activated Cry3A toxin binds to brush border membrane vesicles (BBMV) with a Kd ˜37 nM (Martinez-Ramirez and Real, 1996) and recognizes a 144-kDa binding protein in BBMV prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (Belfiore et al., 1994). Ochoa-Campuzano et al. (Ochoa-Campuzano et al., 2007) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.
Structural differences between Cry3Bb and Cry3Aa toxins could account for the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (Galitsky et al., 2001), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae (Walters et al., 2008). Those authors introduced a chymotrypsin/cathepsin G into domain 1 of Cry3Aa that allowed processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.
Cadherins localized in the midgut epithelium function as receptors for Cry toxins in lepidopteran and dipteran larvae. Sayed et al. (2007) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in midgut tissue of larval stages. The encoded protein is conserved in structure relative to other insect midgut cadherins.
An important Cry1 toxin binding site is localized within the final cadherin repeat (CR) 12 of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctiudae) (Hua et al., 2004; Xie et al., 2005). Unexpectedly, a fragment of Bt-R1 cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (Chen et al., 2007). If the binding residues within cadherin repeat 12 (CR) were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. A fragment of a cadherin from Anopheles gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (Hua et al., 2008).
Terminal cadherin repeats from the Cry1A-binding M. sexta cadherin and the Cry4Ba-binding A. gambiae cadherin were reported to bind toxin and enhance Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae (Chen et al., 2007; Hua et al., 2008).